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An Elegant Analysis of White Spot Syndrome Virus Using a Graphene Oxide/Methylene Blue based Electrochemical Immunosensor Platform

View Article: PubMed Central - PubMed

ABSTRACT

White spot syndrome virus (WSSV) is a major devastating virus in aquaculture industry. A sensitive and selective diagnostic method for WSSV is a pressing need for the early detection and protection of the aquaculture farms. Herein, we first report, a simple electrochemical immunosensor based on methylene blue dye (MB) immobilized graphene oxide modified glassy carbon electrode (GCE/GO@MB) for selective, quick (35 ± 5 mins) and raw sample analysis of WSSV. The immunosensor was prepared by sequential modification of primary antibody, blocking agent (bovine serum album), antigen (as vp28 protein), secondary antibody coupled with horseradish peroxidase (Ab2-HRP) on the GCE/GO@MB. The modified electrode showed a well-defined redox peak at an equilibrium potential (E1/2), −0.4 V vs Ag/AgCl and mediated H2O2 reduction reaction without any false positive result and dissolved oxygen interferences in pH 7 phosphate buffer solution. Under an optimal condition, constructed calibration plot was linear in a range of 1.36 × 10−3 to 1.36 × 107 copies μL−1 of vp28. It is about four orders higher sensitive than that of the values observed with polymerase chain reaction (PCR) and western blot based WSSV detection techniques. Direct electrochemical immunosensing of WSSV in raw tissue samples were successfully demonstrated as a real sample system.

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CV response of different days WSSV infected gill tissues (fresh water crabs; Paratelphusa hydrodomous) modified GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP with 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1.Inset is a plot of ipc of H2O2 reduction reaction vs different day samples. (B) PCR of the different day samples analysed. M-100 bp marker, PC- positive control, NC-negative control, 1–7 lanes denote day-1 to day-7 of gill tissue samples. (C) Western Blot of different days’ gill tissue samples. PC-positive control, NC-negative control, 1 = 1st day gill tissue, 2 = 3rd day gill tissue, 3 = 5th day gill tissue & 4 = 7th day gill tissue. (D) ELISA of time course study gill sample. (E) Plot of the optical density of ELISA measured at 405 nm vs peak current (ipc) of the different day sample.
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f8: CV response of different days WSSV infected gill tissues (fresh water crabs; Paratelphusa hydrodomous) modified GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP with 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1.Inset is a plot of ipc of H2O2 reduction reaction vs different day samples. (B) PCR of the different day samples analysed. M-100 bp marker, PC- positive control, NC-negative control, 1–7 lanes denote day-1 to day-7 of gill tissue samples. (C) Western Blot of different days’ gill tissue samples. PC-positive control, NC-negative control, 1 = 1st day gill tissue, 2 = 3rd day gill tissue, 3 = 5th day gill tissue & 4 = 7th day gill tissue. (D) ELISA of time course study gill sample. (E) Plot of the optical density of ELISA measured at 405 nm vs peak current (ipc) of the different day sample.

Mentions: As a proof of concept and usefulness of this protocol, selective detection of WSSV in raw gill tissues during the course of infection until it reaches moribund stage that usually take about 7 days were examined. Each sample is subjected to triplicate measurements. For convenience, 1st data result was presented in this work. The electrochemical immunosensor preparation procedure follows similar to Fig. 1. Figure 8(A) is a CV response of progression of the WSSV in the infected gill tissue from day 1 to day 7. In parallel, investigations were also carried out using PCR, western blot and conventional ELISA techniques along with positive and negative controls as in Fig. 8(B) to (D) respectively. As seen in Fig. 8(B), the PCR analyses gave specific bands for the 1–7 day samples relating to the qualitative information of the pathogen. There is no significant variation in the band intensities of PCR for the different time duration samples. Similarly, western blot Fig. 8(C) and ELISA Fig. 8(D) analyses results also gave signals only from the day 3 of the post infection. Interestingly, the electrochemical immunosensor results showed specific current signals for all the time duration samples. As can be seen in the Fig. 8(A) and (E), the current signals were increasing proportionately with increase in the post infection time. A plot of immunosensing signal vs post infection time (1–7 day) showed a linear line with slope value of 3.2 μA per day. In addition, a plot of electrochemical signal vs ELISA@ 405 nm OD was found to be linear Fig. 8(E), confirming suitability and reliability of the electrochemical sensor for further routine analysis.


An Elegant Analysis of White Spot Syndrome Virus Using a Graphene Oxide/Methylene Blue based Electrochemical Immunosensor Platform
CV response of different days WSSV infected gill tissues (fresh water crabs; Paratelphusa hydrodomous) modified GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP with 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1.Inset is a plot of ipc of H2O2 reduction reaction vs different day samples. (B) PCR of the different day samples analysed. M-100 bp marker, PC- positive control, NC-negative control, 1–7 lanes denote day-1 to day-7 of gill tissue samples. (C) Western Blot of different days’ gill tissue samples. PC-positive control, NC-negative control, 1 = 1st day gill tissue, 2 = 3rd day gill tissue, 3 = 5th day gill tissue & 4 = 7th day gill tissue. (D) ELISA of time course study gill sample. (E) Plot of the optical density of ELISA measured at 405 nm vs peak current (ipc) of the different day sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5385493&req=5

f8: CV response of different days WSSV infected gill tissues (fresh water crabs; Paratelphusa hydrodomous) modified GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP with 500 μM H2O2 containing pH 7 PBS at v = 10 mV s−1.Inset is a plot of ipc of H2O2 reduction reaction vs different day samples. (B) PCR of the different day samples analysed. M-100 bp marker, PC- positive control, NC-negative control, 1–7 lanes denote day-1 to day-7 of gill tissue samples. (C) Western Blot of different days’ gill tissue samples. PC-positive control, NC-negative control, 1 = 1st day gill tissue, 2 = 3rd day gill tissue, 3 = 5th day gill tissue & 4 = 7th day gill tissue. (D) ELISA of time course study gill sample. (E) Plot of the optical density of ELISA measured at 405 nm vs peak current (ipc) of the different day sample.
Mentions: As a proof of concept and usefulness of this protocol, selective detection of WSSV in raw gill tissues during the course of infection until it reaches moribund stage that usually take about 7 days were examined. Each sample is subjected to triplicate measurements. For convenience, 1st data result was presented in this work. The electrochemical immunosensor preparation procedure follows similar to Fig. 1. Figure 8(A) is a CV response of progression of the WSSV in the infected gill tissue from day 1 to day 7. In parallel, investigations were also carried out using PCR, western blot and conventional ELISA techniques along with positive and negative controls as in Fig. 8(B) to (D) respectively. As seen in Fig. 8(B), the PCR analyses gave specific bands for the 1–7 day samples relating to the qualitative information of the pathogen. There is no significant variation in the band intensities of PCR for the different time duration samples. Similarly, western blot Fig. 8(C) and ELISA Fig. 8(D) analyses results also gave signals only from the day 3 of the post infection. Interestingly, the electrochemical immunosensor results showed specific current signals for all the time duration samples. As can be seen in the Fig. 8(A) and (E), the current signals were increasing proportionately with increase in the post infection time. A plot of immunosensing signal vs post infection time (1–7 day) showed a linear line with slope value of 3.2 μA per day. In addition, a plot of electrochemical signal vs ELISA@ 405 nm OD was found to be linear Fig. 8(E), confirming suitability and reliability of the electrochemical sensor for further routine analysis.

View Article: PubMed Central - PubMed

ABSTRACT

White spot syndrome virus (WSSV) is a major devastating virus in aquaculture industry. A sensitive and selective diagnostic method for WSSV is a pressing need for the early detection and protection of the aquaculture farms. Herein, we first report, a simple electrochemical immunosensor based on methylene blue dye (MB) immobilized graphene oxide modified glassy carbon electrode (GCE/GO@MB) for selective, quick (35 ± 5 mins) and raw sample analysis of WSSV. The immunosensor was prepared by sequential modification of primary antibody, blocking agent (bovine serum album), antigen (as vp28 protein), secondary antibody coupled with horseradish peroxidase (Ab2-HRP) on the GCE/GO@MB. The modified electrode showed a well-defined redox peak at an equilibrium potential (E1/2), −0.4 V vs Ag/AgCl and mediated H2O2 reduction reaction without any false positive result and dissolved oxygen interferences in pH 7 phosphate buffer solution. Under an optimal condition, constructed calibration plot was linear in a range of 1.36 × 10−3 to 1.36 × 107 copies μL−1 of vp28. It is about four orders higher sensitive than that of the values observed with polymerase chain reaction (PCR) and western blot based WSSV detection techniques. Direct electrochemical immunosensing of WSSV in raw tissue samples were successfully demonstrated as a real sample system.

No MeSH data available.


Related in: MedlinePlus